Perturbations in the normal function of transcription factors contribute to the development of a number of diseases. For example, chromosomal translocations that result in mutant transcription factors are associated with the development of several types of cancers. Ewing sarcoma is a pediatric and young adult cancer that results from a translocation that fuses the DNA binding domain of the Friend leukemia virus integration 1 (FLI1) transcription factor to the transactivation domain of the EWS protein, forming a fusion EWS/FLI oncoprotein. Ewing sarcoma cells have a strict dependency on the EWS/FLI oncoprotein, demonstrated by the fact that experimental modulation of EWS/FLI activity (e.g., RNAi knock-down, antisense RNA) results in tumor cell apoptosis, cell cycle arrest, and loss of tumorigenicity. The central role of EWS/FLI in Ewing sarcoma tumorigenesis makes it an attractive drug target; however transcription factor oncoproteins have largely been refractory to conventional drug discovery approaches and are generally considered undruggable. In this proposal, we partner several innovative approaches to identify and subsequently validate small-molecule inhibitors of EWS/FLI. These approaches circumvent many of the hurdles that have hindered the identification of therapies specifically targeting transcription factors. In the first Specific Aim, we will use a well-validated reporter of EWS/FLI activity to screen for small molecules that inhibit EWS/FLI reporter activity, but not a constitutive internal control. Because reporter assays are subject to frequent false-positives, in the second Specific Aim we will use a novel bead-based assay to assess the expression of 123 genes to identify compounds that modulate endogenous EWS/FLI transcriptional activity in Ewing sarcoma cells. Finally, positive hits will be evaluated in the third Specific Aim with a variety of validated tertiary assays to identify potential mechanisms of action. We will work to define and refine the chemical structure of promising lead compounds to identify probes with properties that can be used to develop new therapeutic approaches to selectively target Ewing sarcoma cells. The assays that constitute this discovery pipeline have been optimized for high-throughput screening and are immediately ready for implementation with the MLPCN. Success in these studies will not only identify mature leads for development of Ewing sarcoma targeted therapies, but will also establish a paradigm that can be generalized for identification of inhibitory molecules targeting a variety of transcription factor oncogenes.